The present invention relates to the field of plant molecular biology, in particular to translational control. The inventors herein disclose compositions and methods useful to alter plant gene translation, which methods enable genetic modification of numerous plant processes, such as inducible or constitutive responses to biotic and abiotic stress, or growth pattern adjustment.
In yeasts and mammals, translational control is a reversible, rapid and highly discriminating mechanism of cell regulation. Merrick, W. C. and Hershey, J. W. B. (1996) The pathway and mechanism of eukaryotic protein synthesis. IN: Translational Control. Eds. Hershey, Mathews and Sonenberg. Cold Spring Harbor Press, NY. In these organisms, translational control is critically important in regulation of diverse physiological events including development, differentiation, response to biotic and abiotic stresses and apoptosis. In plants, however, little research has been done on translational control, and virtually nothing is known regarding the impact of translational regulation during plant growth. One of the best characterized translational control mechanisms in yeast and mammals involves the phosphorylation of eIF2xcex1. Although the corresponding proteins have been found in plants, the physiological effects of in planta eIF2xcex1 phosphorylation are not understood.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. For example, in some instances above, the publication was less than one year before the filing date of this patent application. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant at the time of filing, and does not constitute an admission as to the accuracy of the dates or contents of these documents.
The present invention provides methods to affect gene translation in a plant, comprising altering the plant""s inherent eIF2xcex1 activity. In one such embodiment, there are provided methods wherein the alteration has a result selected from the group consisting of: an increase in plant eIF2xcex1 protein amount during a time and in a tissue wherein plant eIF2xcex1 protein is inherently present at a lower amount; an increase in plant eIF2xcex1 protein amount during a time wherein plant eIF2xcex1 protein is inherently present at a lower amount; an increase in plant eIF2xcex1 protein amount in a tissue wherein plant eIF2xcex1 protein is inherently present at a lower amount; an increase in plant eIF2xcex1 protein amount in a tissue wherein plant eIF2xcex1 protein is inherently not present; an increase in plant eIF2xcex1 protein amount during a time wherein plant eIF2xcex1 protein is inherently not present; a decrease in plant eIF2xcex1 protein amount during a time and in a tissue wherein plant eIF2xcex1 protein is inherently present at a higher amount; a decrease in plant eIF2xcex1 protein amount during a time wherein plant eIF2xcex1 protein is inherently present at a higher amount; a decrease in plant eIF2xcex1 in a tissue wherein plant eIF2xcex1 is inherently present at a higher amount.
In a related aspect of the present invention, there are provided methods to increase translation in a plant, comprising increasing plant eIF2xcex1 activity to levels higher than inherently present in the plant. Those methods wherein said increase is by means of genetically-engineering the plant to comprise an expression construct comprising a plant eIF2xcex1 gene are preferred. More preferred are those methods wherein said expression construct is inducible. A most preferred aspect of these embodiments are those methods wherein said expression construct is induced by a condition selected from the group consisting of: pathogen attack; wounding; drought; hypoxia; light; high temperatures; and low temperatures. Methods wherein the plant is selected from the group consisting of: rice; soybean; maize; beet; tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper; melon; cabbage; canola; tulip; orchid and lilly; ornamental plant; turfgrass; horticultural tree; forest tree; conifer; banana tree; grass for hay; lettuce; fruit tree; and bush are preferred.
Also preferred are those methods wherein said plant eIF2xcex1 protein is encoded by a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence which encodes an amino acid sequence which is more than 95% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO 1; SEQ ID NO 3; SEQ ID NO 5; SEQ ID NO 7; SEQ ID NO 9; SEQ ID NO 11; and SEQ ID NO 13; wherein said identity can be determined using the DNAsis computer program and default parameters;
(b) a nucleic acid sequence which is more than 95% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NO 2; SEQ ID NO 4; SEQ ID NO 6; SEQ ID NO 8; SEQ ID NO 10; SEQ ID NO 12; and SEQ ID NO 14; wherein said identity can be determined using the DNAsis computer program and default parameters;
(c) a nucleic acid sequence which encodes an allelic variant of (a).
In other related aspects of the present invention are methods to decrease translation in a plant, comprising decreasing plant eIF2xcex1 activity to levels lower than inherently present in the plant. In this aspect, methods wherein said increase is by means of genetically-engineering the plant to impair inherent plant eIF2xcex1 genes are preferred.
In particular, those methods wherein said impairment is inducible are more preferred, especially with regard to those methods wherein said expression construct is induced by a condition selected from the group consisting of: pathogen attack; wounding; drought; hypoxia; light; high temperatures; and low temperatures. In this aspect, methods are also preferred wherein said plant is selected from the group consisting of: rice; soybean; maize; beet; tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper; melon; cabbage; canola; tulip; orchid and lilly; ornamental plant; turfgrass; horticultural tree; forest tree; conifer; banana tree; grass for hay; lettuce; fruit tree; and bush. Those wherein said plant eIF2xcex1 gene impaired is SEQ ID NO: 1 is particularly preferred.
Also provided are compositions of matter comprising a promoter operably linked to a plant eIF2xcex1 gene. These compositions of matter, wherein said promoter is a constitutive promoter are preferred. Also preferred are compositions of matter wherein said eIF2xcex1 gene is overexpressible and/or overexpressed. Plants comprising these compositions are also provided. Preferred plants are those selected from the group consisting of: rice; soybean; maize; beet; tobacco; wheat; barley; poppy; rape; sunflower; alfalfa; sorghum; rose; carnation; gerbera; carrot; tomato; lettuce; chicory; pepper; melon; cabbage; canola; tulip; orchid; lilly; ornamental plant; turfgrass; horticultural tree; forest tree; conifer; banana tree; grass for hay; lettuce; fruit tree; and bush.
Also provided are knock-out constructs of the eIF2xcex1 gene which are capable of homologous recombination with the wild-type eIF2xcex1 gene, in particular plants comprising a knock-out construct herein.
For the purposes of the present application, the following terms have the following meanings. All other terms have the meaning as generally recognized in the art.
xe2x80x9cAllelic variantxe2x80x9d is meant to refer to a gene that occurs at essentially the same locus (or loci) as the identified reference sequence, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants can also comprise alterations in the 5xe2x80x2 or 3xe2x80x2 untranslated regions of the gene (e.g., in regulatory control regions).
xe2x80x9cInherentxe2x80x9d means that genetic or phenotypic state of being which is present in a plant absent recombinant manipulation of eIF2xcex1 activity. In other words, a plant may be recombinantly altered, physiologically altered (via environmental manipulation) or otherwise altered, provided that the eIF2xcex1 gene present in that particular plant has not been recombinantly modified.
xe2x80x9cKnock-out constructxe2x80x9d means a DNA sequence which has been altered via any known means, for example, deletion, insertion, point mutation or rearrangement, so as to alter (including reduction in function e.g. hindering, and complete deletion) the function of the naturally-occurring gene product, but not so as to alter the ability of the DNA sequence to recombine with the naturally-occurring sequence.
xe2x80x9cOperably-linkedxe2x80x9d means that the promoter is in a position relative to the gene such that the promoter has the ability to effect translation of the gene.
xe2x80x9cPlantxe2x80x9d means any whole plant or plant part, including, but not limited to: monocots and dicots; recombinant or non-recombinant plants; a plant cell culture; or a plant embryo, seed, leaf, flower, stem, root, fruit, etc.